Non-thermosensitive medium for analyzing species in a channel and for minimizing adsorption and/or electroosomosic phenomena

ABSTRACT

An aqueous liquid medium for analyzing, purifying or separating species in an element having walls or for treating the walls of an element. The medium includes at least a polymer consisting of several polymeric segments. The polymer is of the irregular block-copolymer or irregular comb-like polymer type and has on the average at least three junction points between polymeric segments of different chemical or topological nature. The medium may be used in methods for analyzing, purifying or separating species and methods for treating an element to be contacted with a fluid and/or species contained in the fluid during preservation, transport, analysis, purification or separation of the fluid.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/619,878, filed Feb. 11, 2015 (currently pending), which is acontinuation of U.S. application Ser. No. 11/846,670 filed Aug. 29, 2007(now U.S. Pat. No. 8,975,328, issued Mar. 10, 2015), which is acontinuation-in-part application of U.S. application Ser. No. 10/312,537filed Jun. 16, 2003 (now abandoned), which is a National Stageapplication of International PCT Application No. PCT/FR01/002117 filedJul. 2, 2001, which claims priority to French Application No. FR00108528 filed Jun. 30, 2000. U.S. application Ser. No. 11/846,670 alsois a continuation-in-part application of U.S. application Ser. No.10/312,538 filed Jul. 14, 2003 (now abandoned), which is a NationalStage application of International PCT Application No. PCT/FR01/002103filed Jun. 29, 2001, which claims priority to French Application No.00108526, filed Jun. 30, 2000. The subject matter of each of theapplications identified above is incorporated by reference in itsentirety herein.

BACKGROUND OF THE INVENTION

The present invention relates to the field of techniques for analyzing,separating and purifying species, according to which it is necessary tomigrate these species in a fluid known as the “separating medium”.

It is also more particularly directed toward proposing a surfacetreatment solution which is of use in significantly reducing thenonspecific adsorption of species contained in this fluid to the wallsof a channel or of a container containing said fluid.

According to a first aspect, the invention is more particularly directedtoward proposing a separating medium that is suitable for separatingspecies in channels or capillaries, at least one of the dimensions ofwhich is submillimetric, and typically between 1 μm and 200 μm, forexample 20 μm and 200 μm (referred to hereinbelow as microchannels). Theinvention in particular concerns methods for separating or analyzingbiological macromolecules by capillary electrophoresis, bychromatography or by any method used in microchannels (capillaryelectrophoresis and capillary chromatography, microfluid systems, and“lab-on-chips”). The invention is particularly useful in the case ofelectrophoresis.

According to a second aspect the invention also relates moreparticularly to techniques for analyzing or for separating species,according to which it is necessary to transport said species in achannel, while at the same time minimizing the nonspecific interactionsof said species, or of other components of said medium, with the wallsof said channel or more generally with walls or solid elements presentin said channel or said medium. These are in particular methods forseparating or for analyzing biological macromolecules by capillaryelectrophoresis, by chromatography or by any method carried out inmicrochannels (microfluid systems, “laboratories on chips”). Examples ofsuch systems are described, for example, in “Capillary electrophoresisin analytical biotechnology”. Righetti ed., CRC press, 1996, or in J.Cheng et al. (1996), Molecular Diagnosis, 1, 183-200. The invention isof particular use in the case of electrophoresis.

The invention also relates to “hybridization” or “affinity” techniquesin which the aim is to analyze, within a channel or a container, thespecies contained in a sample, as a function of their specific affinityfor ligands contained in said channel or container, or attached to thewalls of said container or said channel, at predetermined positions.

In the context of the invention, the term “nonspecific adsorption” isintended to be used as normally accepted by those skilled in the art, asan interaction of attraction between certain species or impuritiescontained in a sample and the walls of the container and of the channelwhich depends weakly or in an insufficiently controlled manner on thecharacteristics of said species or impurities. In the remainder of thetext, the term “adsorption” or “nonspecific adsorption” will be usedindifferently to denote the latter, as opposed to an interaction ofspecific affinity. The term “affinity” is intended to mean aninteraction between a species and a substrate, the strength of whichdepends strongly on said species and on said substrate, and which, inany event, is sufficient to induce the separation or the identificationof various species as a function of their biological or physicochemicalcharacteristics.

For the purpose of the invention, the terms “solution for treating” and“treatment solution” are equivalent to the term “medium for treating”.

In the text herein below, the expression “microfluid system” will denoteany system in which fluids and/or species contained in a fluid are movedinside a channel or a set of channels, at least one of the dimensions ofwhich is submillimetric, and the term “capillary electrophoresis (CE)”will denote microfluid systems in which the transportation of species isperformed by the action of an electric field.

CE and microfluid systems allow faster separations with higherresolutions than the older methods of gel electrophoresis, do notrequire an anticonvective medium, and their properties have been usedwidely to perform separations of ions in liquid medium. At the presenttime, the vast majority of separations of biological macromoleculesperformed by CE use solutions of interlocked linear water-solublepolymers that have the advantage of being able to be replaced as oftenas necessary.

Many non-crosslinked polymers have been proposed as media for separatingspecies inside a channel, in particular in the context of capillaryelectrophoresis. The choice of the best polymer for a given applicationdepends on several parameters. For example, for the separation ofanalytes as a function of their sizes, it is necessary for the medium topresent the analytes with sufficiently resistant topological obstacles(Viovy et al., Electrophoresis, 1993, 14, 322). This involves theseparating medium being highly interlocked, and thus relatively viscous.It is also necessary for the polymers present in the separating mediumnot to undergo any interactions of attraction with the analytes. Thereason for this is that interactions of this type give rise to aslowing-down of certain analytes, and to additional dispersion (H. Zhouet al. HPCE 2000, Saarbrucken, 20-24 Feb. 2000). Thus, it is well knownthat for DNA sequencing, or for protein separation, poorer results areobtained when the matrix has a more hydrophobic nature.

It has also been proposed in the literature to use copolymers asseparating medium. In Menchen, WO 94/07133, it is proposed to use asseparating medium in capillary electrophoresis, media comprisingcopolymers of block copolymer type which are said to be “regular” sincethey have hydrophilic segments of a selected and essentially uniformlength and a plurality of regularly spaced hydrophobic segments, at aconcentration higher than the overlap concentration between polymers.These media have the advantage of being shear-thinning, i.e. they can beintroduced into a capillary under high pressure, while at the same timepresenting solid topological obstacles in the absence of externalpressure. Unfortunately, the media that may be used according to thisprinciple are difficult to synthesize, which makes them expensive andlimits the type of structures that may be envisaged. Also, thesepolymers are relatively hydrophobic, and their performance qualities forDNA sequencing, for example, are mediocre.

It has also been proposed to use as separating media thermosensitivemedia, the viscosity of which varies greatly during an increase intemperature. This type of medium has the advantage of allowing theinjection of said medium into the capillary at a first temperature in astate of low viscosity, and the separation at a second temperature in astate of higher viscosity that displays good separation efficiency, asis commonly performed in gel electrophoresis, in particular withagarose. Patent applications WO 94/10561 and WO 95/30782 especiallypropose media that allow an easier injection by raising the temperature.In point of fact, said patent applications essentially describemicrogels capable of decreasing in volume at high temperature (thusleading to a dilute solution of discontinuous particles of lowviscosity) and of swelling at low temperature until they entirely fillthe separating channel (thus giving the medium a gelled nature and goodseparating properties). Patent application WO 98/10274 itself proposes amolecular separating medium comprising at least one type of blockcopolymers that is in solution at a first temperature and in a gel-typestate at a second temperature. The media described comprise triblockpolymers of low molecular masses (typically less than 20,000), of thepolyoxyethylene-polyoxypropyiene-polyoxyethylene (POE-Pop-POE) familyand more specifically (POE99-POP69-POE99 in which the indices representthe number of monomers of each block) (trade name “Pluronic F127”). Atlow temperature, the two POE segments at the ends of the triblocksystems are water-soluble and, given the low molecular mass of thecopolymer, the solutions are relatively nonviscous up to a highconcentration. By raising the temperature by about 15-25° C., thecentral POP segment becomes more hydrophobic, and these polymers becomeassociated to form a gel-type state. However, this mechanism presentsseveral drawbacks in electrophoresis. Firstly, it gives rise to a gelstate that has good electrophoretic separating properties only at highpolymer concentrations, of greater than 15 g/100 ml or even 20 g/100 ml,which leads to high friction and long migration times. Moreover, thedependence of the properties as a function of the rate of change oftemperature makes the reproducibility of the results random. Finally,for many applications and in many devices, it is inconvenient, or evenimpossible, to change the temperature between the stage of filling ofthe channel and the separating stage.

In Madabhushi, U.S. Pat. No. 5,552,028, WO 95/16910 and 20 WO 95/16911,it is also proposed to use separating media comprising a screeningmedium and a surface-interaction component consisting of a polymer withwall-adsorption properties, with a molecular mass of between 5,000 and1,000,000, of the disubstituted acrylamide polymer type. These matrices,and more particularly polydimethylacrylamide (PDMA), make it possible toreduce the electroosmosis and in certain applications, for instancesequencing, lead to good separating properties. However, they arerelatively hydrophobic, which limits their performance qualities forcertain applications, for instance DNA sequencing, and is even moreharmful for other applications, for instance protein separation.Moreover, they lead to slow separations.

Consequently, despite the large number of studies and systems proposed,a medium that is optimum for all the various aspects of cost, ofseparation efficiency, of reduction of interactions with the walls andof convenience of use is not available at the present time for all theapplications mentioned above.

In connection to uses of the invention for surface treatment, a majorproblem for all methods involving species within channels is thenonspecific adsorption of said species to the walls of said channels.This problem is particularly exacerbated in the case of channels ofsmall dimensions and of biological macromolecules, the latter oftenbeing amphiphilic.

In the case of analytical methods, the consequence of this phenomenon ofnonspecific adsorption to the walls by species contained in the sampleor the fluid used to analyse said sample, is to delay certain analytesand to create an additional dispersion and therefore a loss ofresolution. This adsorption may also give rise to a contamination of thechannel walls, liable to affect the fluids intended to be subsequentlyintroduced into this channel. Finally, if the analysis to be carried outon the species involves a specific interaction of the species with theseparation medium, as in chromatography, electrochromatography oraffinity electrophoresis methods, or with predetermined areas of thewalls, as in hybridization methods such as “DNA chips” or “proteinchips”, or else with solid walls contained in the channel or container,as in methods of separation by affinity with latexes, these adsorptionphenomena may compete with the desired specific interactions andinterfere with or prevent the analysis.

Another limitation, which concerns more particularly electrokineticseparation methods, is electroosmosis, an overall movement of theseparation medium due to the presence of charges on the walls of thecapillary or of the channel. Since this movement is often variable overtime and is not uniform, it is harmful to the reproducibility of themeasurements and to the resolution. It is due to the charges which maybe present at the surface of the capillary on account of its chemicalstructure, but may also be generated or increased by the adsorption ontothe wall of charged species initially contained in the samples to beseparated, and in particular proteins.

The present invention is more particularly concerned with the inhibitionof these two phenomena, namely adsorption of species to the surfacesand/or electroosmosis.

Methods have already been proposed for combating electroosmosis and/oradsorption of species to surfaces. A first type of method involvestreating the surface of the channel by adsorption of essentially neutralspecies, prior to the actual separation (Wiktorowicz et al.,Electrophoresis, 11, 769, 1990, Tsuji et al., J. Chromatogr. 594, 317(1992)). It has also been proposed to adsorb surface agents with acharge which is opposite to that of the wall, to reinforce the adhesionby electrostatic interactions.

In fact, these methods reduce electroosmosis to a certain degree, butthey are relatively ineffective in preventing the adsorption of complexspecies of high molecular mass, such as, for example, proteins.

A more effective solution consists in irreversibly grafting anessentially neutral polymeric layer, such as acrylamide or polyvinylalcohol, onto the walls, as described, for example, in U.S. Pat. No.4,680,201, or alternatively U.S. Pat. No. 5,502,169 or U.S. Pat. No.5,112,460. Ready-to-use treated capillaries are thus commerciallyavailable. These irreversibly treated capillaries give good reduction ofelectroosmosis for a certain number of separations. Unfortunately, theyhave a limited life span and are expensive.

It has also been proposed to use, in the separation medium, polymerswith properties of adsorption to walls, such as methylcellulose(Hjerten, Chromatographic reviews, 9, 122, 1967) or polyvinylpyrrolidone(Mazzeo at al., Anal. Chem., 63, 2852, 1991). In application WO98/10274, copolymers having affinity with walls of silica and capable ofsignificantly reducing electroosmosis are proposed. The polymersdescribed are triblock polymers of low molecular masses (typically lessthan 20,000), of the polyoxyethylene-polyoxypropylene-polyoxypropylene(POE-POP-POE) family. However, these polymers have a limited range ofapplication. They require a change in temperature between theintroduction into the capillary and the analytical phase, they onlyexert their beneficial effect at high concentrations, and they are alsorelatively hydrophobic, which makes them unsuitable for example for DNAsequencing. In addition, in these various methods of the prior art usingpolymers in the separation medium, the presence of the polymer isaccompanied by a considerable variation in the physical properties, andin particular by a considerable increase in viscosity, which may poseproblems for the introduction of fluid into the channel and for theseparation properties themselves.

In U.S. Pat. No. 5,552,028 already cited above, it is also proposed touse separation media comprising a sieving medium and a surfaceinteraction component consisting of a polymer with properties ofadsorption to walls, having a molecular mass of between 5,000 and1,000,000, of the disubstituted acrylamide polymer type. These matrices,and more particularly polydimethylacrylamide (PDMA), make it possible toreduce electroosmosis and, for some applications, such as sequencing,produce good separation properties. However, they are relativelyhydrophobic, which limits their effectiveness for some applications suchas DNA sequencing, and is even more harmful for other applications suchas protein separation. Moreover, they produce slow separations.

Consequently, although many methods have been proposed for reducingadsorption to walls and/or electroosmosis, they are not found to betotally satisfactory.

BRIEF SUMMARY OF THE INVENTION

One object of the present invention according to its first aspect is,precisely, to propose the use of a family of polymers that isparticularly advantageous as non-thermosensitive liquid separatingmedium for the separation, analysis or purification of species inchannels.

More particularly, a subject of the present invention is anon-thermosensitive liquid medium for analyzing, purifying or separatingspecies inside a channel, and comprising at least one polymer composedof several polymer segments, characterized in that said polymer is ofthe irregular block copolymer type or irregular comb polymer type andhas on average at least three junction points established betweenpolymer segments of different chemical or topological nature.

The object of the present invention is also, according to its secondaspect, to provide a novel family of surface treatment solutions whichare advantageous for minimizing the phenomena of nonspecific adsorptionand of electroosmosis.

More particularly, a subject of the present invention is a solution fortreating the surface of an element intended to be brought into contactwith a fluid and/or species contained in this fluid during thetransport, analysis, purification, separation or conservation of saidfluid, characterized in that said solution comprises at least onepolymer composed of several polymer segments, said polymer being of theblock copolymer or comb polymer type and having on average at leastthree junction points between polymer segments which are chemically ortopologically different in nature.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a, 1b, and 1c are examples of diagrammatic configurations for anirregular sequential block copolymer (1 a), an irregular comb polymer (1b), and an irregular comb copolymer (1 c). The bold lines correspond toone type of chemical nature, and the fine lines to another type ofchemical nature.

FIG. 2 is a control electrophoregram representing the separation of thePharmacia Biotech 50-500 bp sizer, obtained at 50° C. in an ABI 310machine (Perkin Elmer), using an untreated capillary and, as separatingmedium, a 100 mM Na TAPS, 2 mM EDTA, 7M urea buffer, in which isdissolved 5% of a commercial homopolymer of the polyacrylamide type(molecular mass 700,000-1,000,000) the DNA sizes corresponding to thevarious peaks are indicated on the diagram, as number of bases.

FIG. 3 is a control electrophoregram representing a separation underconditions identical to those of FIG. 2, with a “POP6” commercialseparating medium from PE Biosystems. The DNA sizes corresponding to thevarious peaks are indicated on the diagram, as number of bases.

FIG. 4 is an electrophoregram representing a separation under conditionsidentical to those of FIG. 2, with a 100 mM Na TAPS, 2 mM EDTA, 7M ureaseparating medium, in which is dissolved 5% of P(AM-PDMA)-2 combcopolymer described in Example 2. The DNA sizes corresponding to thevarious peaks are indicated on the diagram, as number of bases.

FIG. 5 is a comparison of the resolution calculated between peaksdiffering from one base to 500 bases, obtained at 50° C. in an ABI 310machine (Perkin-Elmer), using as separating medium:

a: a “Pop6” commercial separating medium from PE Biosystems,

b: a 50 mM Na TAPS. 2 mM EDTA, 7M urea buffer, in which is dissolved 5%of linear acrylamide (molecular mass 700,000-1,000,000)

c: the same buffer, in which is dissolved 5% of irregular blockcopolymer according to the invention P(A14-PDMA)-2 described in Example2.

FIG. 6 represents the viscosity of solutions at 3% of linear acrylamide(LPA) and of the copolymers according to the invention poly(AN-PDMA)-1,prepared according to Example 2, poly(AM-PDMA)-2, prepared according toExample 4, and poly(AN-PDMA)-3, prepared according to Example 5.

FIG. 7 represents resolutions obtained during the electrophoreticseparation of DNA, in solutions of linear acrylamide (LPA), ofcommercial polymer (POP5), and of the copolymers according to theinvention poly(AM-PDMA)-1, prepared according to Example 2,poly(AM-PDMA)-2, prepared according to Example 4, and poly(AM-PDMA)-3,prepared according to Example 5, at 3% and 5%.

FIG. 8 is a control electropherogram representing the separation of the50-500 bp sizer. Pharmacia biotech, obtained at 50° C. in an ABI 310device (Perkin-Elmer), using as separation medium a 100 mM Na TAPSbuffer containing 2 mM EDTA and 7 M urea, in which 5% by weight oflinear acrylamide (molecular mass 700,000-1,000,000) is dissolved, in anontreated capillary. The numbers above the peaks indicate the size ofthe corresponding DNA fragment.

FIG. 9 is a control electropherogram representing a separation identicalto that of FIG. 8, in a capillary pretreated for 2 hours with an aqueoussolution containing 3% of triblock copolymer “pluronic F127” (BASF). Thenumbers above the peaks indicate the size of the corresponding DNAfragment.

FIG. 10 is an electropherogram representing a separation identical tothat of FIG. 8, in a capillary pretreated for 2 hours min with anaqueous solution containing 3% of comb polymer of the type having ahydroxyethylcellulose backbone, carrying side chains of the short alkylchain type (NATROSOL PLUS 331, AquaIon). The numbers above the peaksindicate the size of the corresponding DNA fragment.

FIG. 11 is an electropherogram representing a separation identical tothat of FIG. 8, in a capillary pretreated for 2 hours min with anaqueous solution containing 3% of the copolymer according to theinvention “PDMA-NIPAM” described in example 2. The numbers above thepeaks indicate the size of the corresponding DNA fragment.

FIG. 12 is an electropherogram representing a separation identical tothat of FIG. 8, in a nontreated capillary, with copolymer according tothe invention “PDMA-NIPAM” described in example 9 being added to theseparation medium at a concentration by mass of 0.5%. The numbers abovethe peaks indicate the size of the corresponding DNA fragment.

FIGS. 13a and 13b are electropherograms representing a separationidentical to that of FIG. 9:

13 a is polymer “PAM-PDMA-1” described in example 4 being added to theseparation medium at a concentration by mass of 0.5%; the numbers abovethe peaks indicate the size of the corresponding DNA fragment.

13 b is after treatment of the capillary for 2 hours with an aqueoussolution containing 3% of the polymer PAM-PDMA described in example 11.The numbers above the peaks indicate the size of the corresponding DNAfragment.

FIG. 14 is a comparison of the calculated resolution between peaksdiffering by one base to 500 bases, obtained at 50° C. in an ABI 310device (Perkin-Elmer), using as separation medium a 100 mM Na TAPSbuffer containing 2 mM EDTA and 7 M urea, in which 5% of linearacrylamide (molecular mass 700,000-1,000,000) is dissolved, in acapillary initially not treated (“no treatment”), and after pretreatmentof the capillary with an aqueous solution containing 3% of the variouspolymers F127, Natrosol Plus, “PDMA-NIPAM” described in example 9 andPAM-PDMA-1 described in example 11.

FIG. 15 is a comparison of the resolution according to the number ofbase pairs for the separation of a “50-500 bp sizer”(Pharmacia-Amersham), in a solution containing 5% of linearpolyacrylamide exhibiting no wall treatment properties, in a 2 mM EDTA,0.1 M Taps, 7 M urea buffer with addition of 0.5% of the followingpolymers according to the invention: poly(AM-PDMA)-1 (prepared accordingto example 11), poly(AM-PDMA)-2 (prepared according to example 15),poly(AM-PDMA)-3 (prepared according to example 16), poly(DMA-PNIPAM)(prepared according to example 9), and, by way of comparison, withaddition of 0.5% of linear PDMA homopolymer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention more particularly relates to a aqueous liquidmedium, for purifying or separating species inside an element comprisingwalls or for treating the walls of said element, which liquid mediumdoes not display, between its solidification point plus 10° C. and itsboiling point minus 10° C., a variation n in viscosity by a factor of 2or more over a temperature range of 20° C. or less, said mediumcomprising at least one polymer composed of several polymer segments,wherein said polymer is

(a) an irregular block polymer, or

(b) an irregular comb polymer wherein all the segments of a least onetype of chemical or topological nature forming part of the compositionof said comb polymer have a polydispersity of at least 1.5 and whereinthe side branches of said comb polymer have a molecular mass greaterthan 1500, and wherein said polymer has an average of at least threejunction points established between polymer segments of differentchemical or topological nature.

For the purposes of the invention, the term “polymer” denotes a productconsisting of a set of macromolecules and characterized by certainproperties such as molecular mass, polydispersity, chemical compositionand microstructure. The “polydispersity” characterizes the molecularmass distribution of the macromolecules, in the meaning of themass-average familiar to those skilled in the art. The term“microstructure” means the way in which the monomers forming part of thechemical composition of the macromolecules are arranged within thelatter.

According to the invention, the term “liquid” means, as opposed to a“gel”, any condensed medium capable of flowing, whether it is newtonianor viscoelastic.

In the present case, gels derived from the copolymerization of monomersin the presence of difunctional or multifunctional crosslinking agent(s)are excluded from the field of the invention. The reason for this isthat, given their crosslinked state, these gels are solid or elastic andare therefore not liquid. In particular, they are unsuitable forintroduction into a capillary.

The liquid medium of the invention in its different applications,including as a solution treatment for the walls of a container orchannel, and as a separation medium, is nonthermosensitive, i.e. it doesnot display, between its solidification point plus 10° C., and itsboiling point minus 10° C., a sudden change in its viscosity. The term“sudden change” means a variation by a factor of 2 or more, over atemperature range of 20° C. or less.

For the purposes of the invention, the expression “separation method” isintended to cover any method directed toward separating, purifying,identifying or analyzing all or some of the species contained in asample. In this case, the liquid is referred to as the “separatingmedium” and through it pass the species to be separated or at least someof them in the course of the separation process.

The term “species” is generally intended to denote particles, organellesor cells, molecules or macromolecules, and in particular biologicalmacromolecules, for instance nucleic acids (DNA. RNA oroligonucleotides), nucleic acid analogs obtained by synthesis orchemical modification, proteins, polypeptides, glycopeptides andpolysaccharides. In analytical methods, said species are commonlyreferred to as “analytes”.

The invention is particularly advantageous in the case of electrokineticseparation methods.

The term “electrokinetic separation” is intended to cover any methoddirected toward separating all or some of the species contained in amixture by making them migrate in a medium by the action of an electricfield, whether the field exerts its motor action on the analytesdirectly or indirectly, for example by means of a displacement of themedium itself, for instance in electrochromatography, or by means of adisplacement of associated species such as micells, in the case ofmicellar electrochromatography, or by any combination of direct andindirect actions. Any separation method in which said action of theelectric field is combined with another motor action of nonelectricorigin are also considered as an electrokinetic separation methodaccording to the invention. Accordingly, methods of capillaryelectrophoresis or of electrophoresis on “chips” are referred to as“electrokinetic”.

Advantageously, in particular in the case of electrokinetic separations,the liquid will consist of an electrolyte.

For the purposes of the invention, the term “electrolyte” denotes aliquid capable of conducting ions. In the most common case, this mediumis a buffered aqueous medium, for instance buffers based on phosphate,tris(hydroxymethyl)aminomethane (TRIS), borate,N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),histidine, lysine, etc. Numerous examples of buffers that may be used inelectrophoresis are known to those skilled in the art, and a certainnumber of them are described, for example, in Sambrook et al.,“Molecular Cloning: a laboratory manual”, Cold Spring Harbor Lab, NewYork, 1989. However, any type of electrolyte may be used in the contextof the invention, especially aqueous-organic solvents such as, forexample, water-acetonitrile, water-formamide or water-urea mixtures, orpolar organic solvents such as, also by way of example,N-methylformamide. The “sequencing buffer” electrolytes consisting of anaqueous buffer at alkaline pH containing an appreciable proportion ofurea and/or of formamide are found to be particularly useful in thecontext of the invention.

The term “channel” denotes any volume delimited by one or more solidwalls, having at least two orifices and intended to contain a fluid orto have a fluid pass through it.

The term “crosslinked” is intended to mean, as normally accepted bythose skilled in the art, a set of polymers exhibiting between them alarge-scale network of crosslinking points, which confers to this set ofpolymers the properties of a solid or of a gel.

In the present invention, the term “element” is more particularlyintended to denote mainly any channel used for the transport, analysis,purification and separation of a fluid or of species contained in thisfluid, or any container used to conserve a fluid. Also covered underthis definition are solid particles such as beads, for example, liableto be brought into contact with a fluid for the purposes of analysis,separation or purification, in particular by affinity. This definitionalso extends to any element intended to constitute a wall of a channelor of a container used in an operation of transport, analysis,purification, conservation or separation of a fluid or of speciescontained in this fluid, or to be part of said wall. The treatment,using such a solution, of the surface of “DNA chips”, as described, forexample, in “Nature Genetics”, 1999, 21, 1-60, of “protein chips”, ofmicrotitration plates, or more generally of surfaces intended to bebrought into contact with a fluid in a system of analysis or separation,or in a “high throughput screening” system, in particular, falls withinthe scope of the invention. In the description of the invention, theoperation consisting of modifying the properties of the interfacebetween said element and a fluid is indifferently designated by “walltreatment” or “surface treatment”.

According to a preferred variant, useful in applications of theinvention for the treatment of the surface of elements, the polymeraccording to the invention has a chemical composition which is differentfrom that of the materials making up said element. It may thus confer tothe walls of said channel or of said container advantageous propertieswhich are difficult or impossible to obtain in its absence, given thechemical nature of the elements making up the channel or the container.

Advantageously therefore, the polymers used according to the inventionminimize the adsorption of species to the walls and thus improve eitherthe rate of recovery of these species, for example in preparative ormicropreparative systems, or the resolution in analytical methods, orelse avoid the contamination of said walls, in particular in thetransport, analysis or conservation of biological fluids liable tocontaminate said walls or elements.

The invention is particularly advantageous in systems comprising atleast one channel, at least one dimension of which is submillimetric,such as capillary electrokinetic separation systems, microfluid systemsand, more generally, systems for separating species using microchannels,microcontainers or nanocontainers.

According to one preferred variant, the polymers according to theinvention exhibit on average at least four junction points, preferably anumber of junction points of between 4 and 100 and more preferably anumber of junction points of between 4 and 40.

The term “junction point” means a point connecting either two polymersegments of significantly different chemical nature, for instance in thecase of a block copolymer, or a point of crosslinking between more thantwo polymer segments of identical or different chemical nature, forinstance in comb polymers. By way of example, a comb polymer bearingthree side branches comprises three junction points and seven separatepolymer segments. Similarly, a sequential block copolymer of A-B-A-Btype comprises three junction points and four separate polymer segments.

For the purposes of the invention, the terms “polymer segment” and“segment” denote a set of monomers linked together in a linear andcovalent manner, and belonging to a given type of chemical composition,i.e. having specific overall physicochemical properties, in particularas regards the solvation, the interaction with a solid wall, a specificaffinity toward certain molecules, or a combination of these properties.

An example of a polymer segment for the purposes of the invention isgiven by the sequence, within a copolymer, of monomers that are allidentical (homopolymer segment), or a copolymer that has no significantcomposition correlation over distances of more than a few monomers(segment of random copolymer type).

The polymer according to the invention is composed of several“different” polymer segments. Two polymer segments that differ in theirchemical nature and/or their topology, i.e. the spatial distribution ofthe segments relative to each other, for example skeleton as opposed toside branch, are different for the purposes of the invention.

According to a first preferred variant, the polymers according to theinvention are of the irregular block copolymer type.

For the purposes of the invention, the term “block copolymer” denotes acopolymer consisting of several polymer segments linked togethercovalently, and belonging to at least two different types of chemicalcomposition. Thus, two adjacent polymer segments within a linear blockcopolymer are necessarily of significantly different chemical nature.The block copolymer is defined by the fact that each of the segmentscomprises a sufficient number of monomers to have within the separatingmedium physicochemical properties, and in particular in terms ofsolvation, that are comparable to those of a homopolymer of the samecomposition and of the same size. This is in contrast with a randomcopolymer, in which the various types of monomer follow each other in anessentially random order, and give the chain locally overall propertiesthat are different from those of homopolymers of each of the speciesunder consideration. The size of the homopolymer segments required toobtain this block nature may vary as a function of the types of monomersand of the electrolyte, but it is typically a few tens of atoms alongthe skeleton of said segment. It should be noted that it is possible tomake a block copolymer within the meaning of the invention, in which allor some of the segments themselves consist of a copolymer of randomtype, insofar as it is possible to distinguish within said blockcopolymer polymer segments of size and of difference in chemicalcomposition that are sufficient to give rise from one segment to anotherto a significant variation in the physicochemical properties, and inparticular in the solvation. In particular, in order to be considered asa “polymer segment” within the meaning of the invention, a portion ofpolymer must comprise along its skeleton at least 10 atoms.

According to one preferred mode of this variant, the polymer accordingto the invention is of the irregular sequential block copolymer type.

For the purposes of the invention, the term “sequential block copolymer”means a block copolymer composed of polymer segments belonging to atlest two different chemical types, linked together in a linear manner.

According to a second preferred variant, the polymer according to theinvention is of the irregular comb polymer type.

In this case, one preferred variant of an irregular comb polymerconsists in displaying segments of at least one type of chemical ortopological nature forming part of the composition of said comb polymerhaving a polydispersity of at least 1.5 and side branches of said combpolymer having a molecular mass greater than 1500.

For the purposes of the invention, the term “comb polymer” denotes apolymer having a linear skeleton of a certain chemical nature, andpolymer segments known as “side branches”, of identical or differentchemical nature, which are also linear but significantly shorter thanthe skeleton, and are covalently attached to said skeleton via one oftheir ends. In a comb polymer, the polymer segments constituting theskeleton and those constituting the side branches differ in theirtopological nature. If the polymer segments constituting the sidebranches of the comb polymer and those constituting its skeleton alsodiffer in their chemical nature, the polymer simultaneously has thecharacteristic of a “comb polymer” and that of a “block copolymer”. Suchpolymers, which are known as “comb copolymers”, constitute a subset ofcomb polymers and can, of course, be used in the context of theinvention.

Needless to say, the combined use of block copolymer(s) and combpolymer(s) in a medium in accordance with the invention may beenvisaged.

The number of polymer segments of a given chemical or topological typepresent in the polymers according to the invention is understood asbeing an average value, it being understood that it is always a matterof a population of a large number of molecules having in said numbers acertain polydispersity.

In the present description and unless otherwise mentioned, all themolecular masses and also all the averages for all the chains or all thepolymer segments, for instance the average molecular mass, or theaverage number of atoms along the skeleton, the number of junctionpoints, or the average number of grafts in the case of a comb polymer,are understood as being mass averages within the usual meaning ofpolymer physics.

All the polymers under consideration according to the invention, namelyblock copolymers or comb polymers, also have the advantageouscharacteristic of being of irregular type. According to one aspect ofthe invention, all the segments of at least one type of chemical ortopological nature forming part of their composition have apolydispersity of at least 1.5 and preferably greater than 1.8.

The polydispersity of a type of polymer segment forming part of thecomposition of a polymer according to the invention is understood asbeing the average value of the molecular mass of said segments, takenover all the segments of this type forming part of the composition ofsaid polymer (mass average within the usual meaning of polymerphysicochemistry).

Another preferred variant of an irregular comb polymer consists indisplaying a polydispersity of the segments of the skeleton includedbetween two side branches of at least 1.5 and preferably greater than1.8.

In another preferred embodiment, the segments of each of the types ofchemical or topological nature forming part of the composition of thepolymers according to the invention have a polydispersity of at least1.5 and preferably greater than 1.8.

According to one preferred embodiment, the polydispersity of thepolymers according to the invention is greater than 1.5 and preferablygreater than 1.8.

The length and number of the different polymer segments present in thecomb polymers or the copolymers used in the media according to theinvention, and also the chemical nature thereof, may vary significantlyin the context of the invention, and the properties of said media maythus be varied widely depending on the desired application, as will beshown more specifically in the description of the implementationexamples.

According to one preferred embodiment, at least one segment of thepolymer according to the invention is of hydrophilic nature.

According to one preferred embodiment, the polymers according to theinvention have a molecular mass (massaverage) greater than 50,000,preferably greater than 300,000, more preferably greater than 1,000,000and better still greater than 3,000,000.

According to one preferred embodiment, said polymers according to theinvention show within the separating medium significant affinity for thewalls of said channel.

One particularly preferred mode consists in presenting within thepolymer according to the invention at least one type of polymer segmentshowing, within the separating medium contained in a channel with walls,or within the fluid in which it is carried for the purpose of treatingthe wall of an element, specific affinity for the wall, and at least onetype of polymer segment showing in said medium less or no affinity forthe wall.

The presence of polymer segments of this type allows the mediumaccording to the invention to reduce the adsorption of species onto thewalls of the channel and/or the electroosmosis.

The polymers according to the invention of the type containing polymersegments of at least one type showing within the separating mediumspecific affinity for the wall, have, on account of the presence of aplurality of segments of this type, and on account of the relativelyhigh average molecular mass of said segments, a high adsorption energy,and thus reduce the electroosmosis in a long-lasting manner. Moreover,since the polymers according to the invention also comprise in theirstructure polymer segments that show in said medium less or no affinityfor the wall, they avoid an excessively hydrophobic nature that isharmful for resolution, and can more efficiently repel the analytes fromthe walls.

Typically, types of polymer segments that show no affinity for the wallconsist of polymers that show good solubility in the separating medium.However, there may be polymers which are soluble in said medium, butwhich nevertheless show therein particular affinity for a wall. When theseparating medium is an aqueous solution, segments with no affinity forthe wall are typically highly hydrophilic segments. On the other hand,segments with affinity are relatively nonhydrophilic, or evenhydrophobic. Needless to say, other more specific types of affinity maybe used, depending on the nature of the wall and that of the separatingmedium or that of the fluid in which said polymer is carried for thepurpose of treating the wall of an element.

Copolymers that are optimized for performing the invention areespecially those in which all the segments that have specific affinityfor the wall represent between 2% and 80% by mass and preferably between5% and 50% or 5% and 30% of the total average molar mass of saidcopolymers, or between 3% and 85% or 3% and 90% and preferably between5% and 50%, or 5% and 60% of the total composition of the copolymers interms of number of moles of monomers.

Another preferred embodiment, which is particularly advantageous whenthe analytes are biological macromolecules, consists in using polymersaccording to the invention that also show specific affinity for one ormore analytes.

This affinity may be obtained by incorporating into the structure ofsaid polymers polymer segments capable of showing specific affinity forcertain analytes. Such polymer segments may consist, for example, and ina nonexhaustive manner, of a predetermined sequence of differentmonomers, for instance a polynucleotide or a polypeptide. This affinitymay also be obtained by combining with the polymer according to theinvention a native or denatured protein, a protein fraction or a proteincomplex, or alternatively an acidic or basic function, and/or a functionof Lewis acid or Lewis base type.

According to another embodiment, particularly useful for surfacetreatments, the copolymers which are optimized for use of the inventionare in particular those in which all of the segments exhibiting aparticular affinity with the wall represent between 2 and 25% by mass,preferably between 5 and 15%, of the average total molar mass of saidcopolymers, or between 3 and 30%, and preferably between 5 and 20% ofthe total composition of the copolymers in number of moles of monomers.

As illustrations of the various structures that may be adopted by thecopolymer according to the invention, mention may be made mostparticularly of those in which all or some of said copolymer is:

-   -   in the form of irregular sequential block copolymers. In this        case, one preferred variant consists in alternating, along the        polymer, segments with specific affinity for the wall, and        segments with reduced or no affinity for the wall. It may also        be envisaged to alternate, along the polymer, segments showing        specific affinity for certain analytes, and segments showing        reduced or no affinity for said analytes;    -   in the form of irregular comb copolymers. In this case, one        preferred variant is characterized in that said polymers are in        the form of comb polymers whose skeleton consists of several        polymer segments that show specific affinity with the wall, and        the side branches of which consist of polymer segments showing        reduced or no affinity for the wall, or comb polymers whose side        branches consist of polymer segments showing specific affinity        for the wall, and whose skeleton consists of polymer segments        showing reduced or no affinity for the wall. These polymers may        also be in the form of comb polymers, certain side branches of        which consist of polymer segments showing specific affinity for        certain analytes, and the skeleton of which consists of polymer        segments showing reduced or no affinity for these analytes.

Needless to say, systems in which several types of preferred variantsabove are combined together, either by combining polymer segments ofmore than two different types, or in the form of a mixture of differentcopolymers, also fall within the scope of the invention. It is thuspossible, for example, to combine within a copolymer according to theinvention polymer segments showing affinity for the wall, polymersegments or groups showing specific affinity for certain analytes, andpolymer segments showing no specific affinity either for the walls orfor the analytes. It is also possible, again by way of example, tocombine in a medium according to the invention block copolymerscomprising polymer segments showing affinity for the wall and polymersegments showing no specific affinity either for the walls or for theanalytes, and polymers comprising polymer segments or groups showingspecific affinity for certain analytes, and polymer segments showing nospecific affinity either for the walls or for the analytes.

In one preferred mode of the invention, all of the polymer segments of agiven type of chemical or topological nature have on average along theirskeleton a number of atoms of greater than 75, and more preferablygreater than 210, or have a molecular mass of greater than 1,500 andpreferably greater than 4,500.

According to an even more preferred embodiment, the various types ofsegments have along their skeleton an average number of atoms of greaterthan 75, and more preferably greater than 210, or have a molecular massof greater than 1,500 and preferably greater than 4,500.

According to one preferred embodiment, the invention also relates to aseparating medium consisting in a liquid in which at least one polymerin accordance with the invention is dissolved to a proportion of from0.1% to 20% and preferably from 1% to 6% by weight. It is particularlyadvantageous, for implementing the invention, to use block copolymers orcomb homopolymers in which at least one of the types of segmentsconsists of a polymer chosen from polyethers, polyesters, for instancepolyglycolic acid, soluble random homopolymers and copolymers of thepolyoxyalkylene type, for instance polyoxypropylene, polyoxybutylene orpolyoxyethylene, polysaccharides, polyvinyl alcohol,polyvinylpyrrolidone, polyurethanes, polyamides, polysulfonamides,polysulfoxides, polyoxazoline, polystyrene sulfonate, and substituted orunsubstituted acrylamide, methacrylamide and allyl polymers andcopolymers.

As representatives of the types of polymer segments showing, in anaqueous separating medium, little or no affinity with the walls, mentionmay be made most particularly of polyacrylamide and polyacrylic acid,polyacryloylaminopropanol, water-soluble acrylic and allylic polymersand copolymers, dextran, polyethylene glycol, polysaccharides andvarious cellulose derivatives such as hydroxyethylcellulose,methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose ormethylcellulose, polyvinyl alcohol, polyurethanes, polyamides,polysulfonamides, polysulfoxides, polyoxazoline, polystyrene sulfonate,and also polymers bearing hydroxyl groups, and all the random copolymersof the derivatives mentioned above.

Needless to say, other polymer segments that are soluble in theseparating medium may be used according to the invention, as a functionof the nature of said fluid and of that of the walls of the channel, theparticular application and the ease of introducing them into a blockpolymer of the desired structure.

As representatives of the polymer segments, which may or may not besoluble in aqueous solvents, and which may show therein particularaffinity for the walls, mention may be made of dimethylacrylamide,acrylamides N-substituted with alkyl functions, acrylamidesN,N-disubstituted with alkyl functions, allyl glycidyl ether, copolymersof the above acrylic derivatives with each other or with other acrylicderivatives, alkanes, fluoro derivatives, silanes, fluorosilanes,polyvinyl alcohol, polymers and copolymers involving oxazolinederivatives, and also in general polymers that have a combination ofcarbon-carbon bonds, ether-oxide functions and epoxide functions, andalso all the random copolymers of these compounds.

Many types of polymer segments may be chosen to make up the polymersegments constituting a polymer according to the invention, as afunction of the envisaged electrolyte, from all the types of polymersknown to those skilled in the art, in particular from those soluble inaqueous medium. Reference may thus be made to the book “PolymerHandbook” Brandrupt & Immergut, John Wiley, New York.

Another preferred embodiment, which is particularly advantageous whenthe species to be separated are biological macromolecules, consists inusing copolymers according to the invention which also exhibit adifferent affinity with different analytes.

This affinity may be obtained by integrating into the structure of saidpolymers segments capable of exhibiting an affinity specific for certainspecies to be separated. Such segments may, in a nonexhaustive manner,consist, for example, of a predetermined sequence of different monomers,such as a polynucleotide or polypeptide. This affinity may also beobtained by associating with the polymer according to the invention anative or denatured protein, a protein fraction or a protein complex, oralternatively an acid or basic function and/or a function such as anacid or base in the Lewis sense.

According to this variant, it is possible to envision either alternatingalong a linear block copolymer segments exhibiting a specific affinitywith certain analytes and segments exhibiting less or zero affinity withsaid analytes, or introducing into a comb copolymer polymer segmentsexhibiting an affinity specific for certain analytes, either in the formof additional side branches or of subsidiary segments in their backbone.

The polymers according to the invention may be natural or synthetic.According to one preferred variant for the variety and control that itallows with regard to the microstructure, the polymers according to theinvention are synthetic polymers.

The following are most particularly suitable for the invention:

-   -   copolymers of the comb copolymer type, the skeleton of which is        of dextran, acrylamide, acrylic acid, acryloylaminoethanol or        (N,N)-dimethylacrylamide type and onto which are grafted side        segments of acrylamide, substituted acrylamide or        (N,N)-dimethylacrylamide (DMA) type, or of the DMA/allyl        glycidyl ether (AGE) copolymer type, or alternatively of        homopolymer or copolymer of oxazoline or of oxazoline        derivatives;    -   non-thermosensitive copolymers of the irregular sequential block        copolymer type having along their skeleton an alternation of        segments of polyoxyethylene type and of segments of        polyoxypropylene type, or an alternation of segments of        polyoxyethylene type and of segments of polyoxybutylene type, or        more generally an alternation of segments of polyethylene and of        segments of polyether type that are appreciably more hydrophobic        than polyoxyethylene;    -   copolymers of the irregular sequential block copolymer type        having along their skeleton an alternation of segments of        acrylamide, acrylic acid, acryloylaminoethanol or        dimethylacrylamide type, on the one hand, and segments of        (N,N)-dimethylacrylamide (DMA) type, or of DMA/allyl glycidyl        ether (AGE) copolymer type, or alternatively of homopolymer or        copolymer of oxazoline or of oxazoline derivatives;    -   polymers of the irregular comb polymer type, the skeleton of        which is of agarose, acrylamide, substituted acrylamide, acrylic        acid, acryloyl-aminoethanol, dimethylacrylamide (DMA), or allyl        glycidyl ether (AGE) polymer type, of DMA/AGE random copolymer        type, of oxazoline and oxazoline derivative, of dextran, of        methylcellulose, of hydroxyethylcellulose, of modified        cellulose, of polysaccharide or of ether oxide type, and onto        which are grafted side segments of agarose, acrylamide,        substituted acrylamide, acrylic acid, acryloylaminoethanol,        dimethylacrylamide (DMA), or allyl glycidyl ether (AGE) polymer        type, of DMA/AGE random copolymer type, of oxazoline and        oxazoline derivative, of dextran, of methylcellulose, of        hydroxyethylcellulose, of modified cellulose, of polysaccharide        or of ether oxide type;    -   copolymers of the irregular comb copolymer type, the skeleton of        which is of the acrylamide, substituted acrylamide, acrylic        acid, acryloylaminoethanol, dimethylacrylamide (DMA), or allyl        glycidyl ether (AGE) polymer type, of DMA/AGE random copolymer        type, of oxazoline and oxazoline derivative, of dextran, of        agarose, of methylcellulose, of hydroxyethylcellulose, of        modified cellulose, of polysaccharide or of ether oxide type,        and bears short-chain hydrophobic side segments such as alkyl        chains, aromatic derivatives, fluoroalkyls, silanes or        fluorosilanes.

It should also be noted that, in most applications, it is preferable touse a polymer according to the invention that is essentially neutral.However, it may be useful for certain applications, and in particular toavoid the adsorption of species containing both charges and hydrophobicportions, to select a polymer according to the invention that isdeliberately charged, preferably opposite in charge to that of saidspecies.

The copolymers according to the invention are advantageous because oftheir ability to combine properties belonging to polymers which arechemically different in nature, which cannot always be brought togetherin a homopolymer or a random copolymer. Thus, they allow more flexibleadaptation of the chemical nature of the copolymer, as a function,firstly, of the chemical nature of the fluid and, secondly, of thechemical nature of the wall of the channels or containers. They are thusparticularly advantageous in applications using channels or containersconsisting of polymers or elastomers such as PDMS(polydimethylsiloxane), PMMA poly(methyl methacrylate), polycarbonate,polyethylene, polypropylene, polyethylene terephthalate, polyimide,polycyclohexane, polyurethanes or organic materials such as ordinaryglass, borosilicate glass. Pyrex, molten silica, silicon oxide,ceramics, silicon, diamond, zirconium or semiconductors. Moreover, thepolymers according to the invention have the unique property of beingable to have, on each polymer, a considerable number of polymer segmentsexhibiting a significant affinity with the wall, which allows a largeenergy of absorption, and therefore a long-lasting reduction ofelectroosmosis, while at the same time also containing a considerablenumber of loops not exhibiting affinity with the walls, which may serveto avoid the adsorption of the species.

As regards the preparation of the copolymers used according to theinvention, it may be carried out by any conventional polymerization orcopolymerization technique. The choice of preparation method isgenerally made by taking into account the structure desired for thecopolymer, i.e. comb or linear structure, and the chemical nature of thevarious blocks of which it is made.

As representatives of these preparation variants, mention may be mademost particularly of processes according to which said copolymers areobtained by:

-   -   polycondensation, ionic or free-radical polymerization or        copolymerization of identical or different monomers, of        identical or different macromonomers, or of a mixture of        identical or different monomers and macromonomers, or    -   by grafting several polymer segments onto a linear or branched        polymer skeleton of identical or different chemical nature.

Preferably, all or some of the copolymers used according to theinvention are obtained by

-   -   a: copolymerization of monomers and macromonomers comprising a        reactive function at at least one of their ends, or    -   b: copolymerization of macromonomers comprising at least one        reactive function in their structure.

For the purposes of the invention, the term “reactive function” means agroup that allows the molecule bearing this group to be incorporatedinto the macromolecule during the copolymerization reaction withoutinterrupting said copolymerization.

With the aid of the rules and preferred modes listed above, a personskilled in the art is capable of preparing the copolymers in accordancewith the invention, by adapting the structure, the nature and the modeof preparation of said polymers as a function of the desired separationproperties for one application or another.

A subject of the present invention is also a process for separating,analyzing and/or identifying species contained in a sample,characterized by performing the following steps:

a/ filling the channel of a separating device with separating mediumaccording to the invention,

b/ introducing said sample containing said species into one end of saidchannel,

c/applying an external field intended to move certain species containedin the sample, especially an electric field, and

d/recovering or detecting the passage of said species at a point alongthe channel that is different from the point of introduction of thesample.

In one preferred variant, it is not necessary to change the temperaturebetween the capillary filling stage and the analysis stage.

Depending on the particular applications, the separating medium maycontain, besides the polymers according to the invention, otherelements, and in particular components that interact with the species orthe walls. Many elements of this type are known to those skilled in theart.

In the present case, it is possible to combine in the separating mediumpolymers of the irregular block copolymer type, and other polymerscapable of interacting with analytes either by steric interaction or byaffinity, in order to improve the performance qualities compared withthose obtained with the polymer according to the invention used alone.In this case, polymers that are more particularly preferred according tothe invention are those having a mass fraction of polymer segmentsshowing specific affinity for the wall that is greater than when thesepolymers are used alone. This fraction may be between 20% and 80%.

A subject of the present invention is also the use of a separatingmedium according to the invention for separating, purifying, filteringor analyzing species chosen from molecular or macromolecular species,and in particular biological macromolecules, for instance nucleic acids(DNA, RNA or oligonucleotides), nucleic acid analogs obtained bysynthesis or chemical modification, proteins, polypeptides,glycopeptides and polysaccharides, organic molecules, syntheticmacromolecules or particles such as mineral particles, latices, cells ororganelles.

In the case of electrophoresis analysis methods, the invention isparticularly useful for DNA sequencing, for which it allows minimumbandwidths to be obtained. It is also particularly favorable forseparating proteins, proteoglycans, or cells, for which the problems ofadsorption onto the wall area particular handicap and particularlydifficult to solve.

According to one aspect of the invention, the separation or analyzing ofspecies of the process according to the invention involvesdiscrimination of said species by affinity or molecular hybridization.

Advantageously, the claimed medium may be used in a channel of which atleast one dimension is of submillimetric size.

As regards the apparatus, the claimed medium is particularlyadvantageous for microfluidic systems, since it makes it possible, bymeans of an optimum choice of the various types of blocks within thepolymers, to combine blocks that show good affinity for the surface ofthe channel in order to obtain a long-lasting treatment, and blocks thatshow good repulsion for the species to be separated, irrespective ofsaid species and of the chemical nature of said channel.

The media according to the invention and the separation methods usingthese media are particularly advantageous for electrophoretic separationand diagnostic applications, gene typing, and high-throughput screening,quality control, or for detecting the presence of genetically modifiedorganisms or pathogens in a product, or detecting the presence ofdangerous material in an object or device.

In point of fact, the polymers of which the separating medium underconsideration in the context of the present invention is composed arefound to be advantageous in several respects.

Firstly, their capacity to display “block polymer” nature allows them tocombine properties belonging to polymers of different chemical nature,and that cannot always be united in a homopolymer or a random copolymer.They thus make it possible to more flexibly adapt the chemical nature ofthe separating medium, firstly as a function of the species to beseparated, and secondly as a function of the chemical nature of thechannels in which the separation is performed. They are thusparticularly advantageous both in applications using channels consistingof polymers or elastomers such as PDMS (polydimethylsiloxane), PMMA(polymethyl methyacrylate), polycarbonate, polyethylene, polypropylene,polyethylene terephthalate or polyimide, or of mineral materials such asglass, ceramics, silicon, stainless steel or titanium, and in moretraditional applications using channels whose walls are made of fusedsilica.

Compared with the block copolymers of the prior art, the polymersaccording to the invention also show superior performance qualities interms of resolution, which is most probably associated with theirirregular nature, i.e. the polydispersity of the polymer segmentsforming part of the polymers according to the invention. Thischaracteristic is particularly surprising, since the set of blockcopolymers used in the prior art deliberately involves copolymerscontaining regularly spaced segments and/or having a selected andessentially uniform length (i.e. low polydispersity). Thispolydispersity of the segments, in the polymers according to theinvention, also shows advantages in terms of cost and flexibility informulating, since polymers comprising such polymolecular segments arenot only more efficient, but also easier to prepare. In particular, theymay be prepared with high molecular masses.

In the applications for which a reduction of electroosmosis orinteraction of species with the wall is desired, the polymers accordingto the invention have, on account of the presence in their structure ofa large number of polymer segments that show significant affinity forthe wall, high adsorption energy and can thus reduce the electroosmosisand the adsorption of species in a long-lasting manner.

Finally, it is also very likely that the combination of a linearskeleton and of a plurality of junction points gives the separatingmedia according to the invention some of the properties of gels at thelocal scale, which is beneficial in terms of separation efficiency,while at the same time conserving them at the large scale, and inparticular as regards the flow properties, properties that arecomparable with those of linear polymers.

Depending on the applications, the chemical nature of the capillary forwhich the surface treatment solution according to the invention, alsocalled medium for treating the walls of the channel, is intended and theparticular choice of polymer according to the invention used for thetreatment, said solution may comprise, as a basis for dissolving thecopolymers according to the invention, an aqueous (preferably buffered)solution, an organic solvent, an aqueous-organic solvent or anelectrolyte.

In a preferred variant, the polymers contained in the surface treatmentsolutions according to the invention attach to the solid walls orsurfaces thanks to at least physical adsorption without establishing acovalent bond.

According to another preferred variant, the polymers contained in thesurface treatment solutions according to the invention attach to thesolid walls or surfaces thanks to at least one or more covalent bonds.

Using the preferred rules and modes stated above, those skilled in theart are capable of preparing polymers in accordance with the invention,by adapting the structure, the nature and the method of preparation ofsaid polymers as a function of the desired properties for oneapplication or another.

A subject of the invention is also a process for treating the surface ofan element, in particular to avoid the phenomena of electroosmosisand/or of nonspecific adsorption of species capable of manifestingthemselves at this surface when it is brought into contact with a fluid,and/or of species contained in this fluid.

More precisely, it is a process for treating the surface of an elementintended to be brought into contact with a fluid and/or speciescontained in this fluid during the transport, analysis, purification,separation and/or conservation of said fluid, comprising bringing saidelement into contact with at least one noncrosslinked polymer of theblock copolymer or comb polymer type, having on average at least threejunction points between polymer segments chemically or topologicallydifferent in nature and more particularly bringing said element intocontact with at least one medium according to the invention.

According to one aspect of the present invention, the fluid is abiological fluid, a fluid containing or liable to be contamined withorganic or biological products, or a fluid containing or liable to becontamined with live organisms.

For the definition of the polymer and of the element, reference will bemade to the preceding description.

According to a preferred variant of the invention in its surfacetreatment aspect, the polymer is used in the form of an aqueous solutionof polymer as claimed and preferably containing said polymer at aconcentration of between 0.01% and 20%, and more preferentially between0.1 and 5% by mass.

According to a first embodiment, the process comprises treating theelement, prior to its use, with a treatment solution in accordance withthe invention.

When the treatment is carried out for the purpose of a transport orconservation operation, this solution has a composition different fromthat of the fluid intended to be transported or conserved. When thetreatment is carried out for the purpose of a separation application,this solution has a composition different from that of the separationmedium. The treatment is obtained by leaving said solution in contactwith the walls for the necessary period of time. Depending on theapplication and the embodiment, this period of time may be veryvariable, ranging from a fraction of a second to several hours, or even,for the most difficult applications, several days. This solution is thenremoved from the channel or container, prior to or simultaneously withthe filling thereof with said fluid. According to this variant, saidfluid does not, itself, contain the polymers according to the invention.Thus, the latter remain present in the channel or the container only ina form adsorbed to the walls, and do not contribute to modifying theproperties of said fluid. In particular, they do not significantlyincrease its viscosity. Depending on the application, this treatment maybe renewed between each transport or separation operation, or, on theother hand, after a given number of separations or else when adegradation of the properties is noted, making it necessary.

According to a preferred variant of the invention, the bringing of saidsurface treatment solution into contact with the surface of the elementwith respect to which a reduction of the nonspecific adsorption or theelectroosmosis is desired may be followed by a treatment intended toreinforce the action of said solution, such as, by way of nonlimitingexample, thermal treatment, treatment by radiation (light radiation,ultraviolet radiation, X rays, gamma rays, etc.), drying of the wall, orincubation thereof in the presence of a liquid different from saidsolution.

However, this treatment is not always necessary, and some polymersaccording to the invention which are part of the composition of thesurface treatment solutions of the invention are capable of effectivelyminimizing the nonspecific adsorption and the electroosmosis withoutsubsequent treatment.

According to a preferred embodiment, in particular if many separationsare carried out between two surface treatments with a solution accordingto the invention, the surface of the element may be “regenerated” beforethe treatment, with a solution intended to clean off the wall theimpurities adsorbed in the course of the separations. Such treatmentsare known to those skilled in the art and may advantageously comprisewashing with an acid solution, with an alkaline solution, with asolution of detergent, with an organic solvent, or with a combination ofthese methods.

According to a second embodiment, the process claimed comprises theaddition of said polymer to the fluid which must be transported,analyzed, purified, separated and/or conserved.

According to this second embodiment, the copolymers which characterizethe surface treatment solutions according to the invention arepreferably introduced directly into the fluid transported, conserved orused as a separation medium, at a concentration sufficiently low so asnot to significantly modify, moreover, the other customary properties ofsaid fluid, and in particular without increasing its viscosity more than2-fold, relative to the same fluid in the absence of said polymers.According to an even more preferred variant, the polymers according tothe invention do not modify the viscosity of said fluid more than1.5-fold.

As regards the fluid into which the polymer according to the inventionis directly introduced, it may advantageously contain, besides thepolymers according to the invention, other elements, and in particularcomponents which interact with the species either by steric interactionor by affinity, and which are capable of inducing between one another atotal or partial separation of these species. Many components of thistype, such as hydrophilic linear polymers, micelles, surfactants orchiral compounds are known to those skilled in the art.

Of course, the present invention extends to any process of separation,filtration, analysis and/or purification involving the use of theclaimed process. These processes of filtration, separation, analysisand/or purification are partly identified below.

The present invention also relates to an element, preferably channel,container or particles, or any element intended to constitute a wall ofa channel or of a container used in an operation of transport, analysis,purification, conservation or separation of a fluid or of speciescontained in this fluid, or intended to form part of said wall, treatedwith the surface treatment solution claimed.

Such elements may be used for the separation, purification, filtrationor analysis of species chosen from molecular or macromolecular species,and in particular biological macromolecules such as nucleic acids (DNA.RNA, oligonucleotides), nucleic acid analogs obtained by chemicalmodification or synthesis, proteins, polypeptides, glycopeptides andpolysaccharides, organic molecules, synthetic macromolecules orparticles such as mineral particles, latex particles, cells ororganelles.

The elements treated according to the invention are also of particularuse for DNA sequencing insofar as they make it possible to obtainminimum bandwidths. Similarly, they are found to be suitable forseparating proteins, proteoglycans or cells, for which it is known thatproblems of adsorption to the wall are particularly bothersome andparticularly difficult to solve.

However, the possibility offered by the invention of greatly varying thechemical nature of the surface is also advantageous for otherapplications.

The surface treatment solutions according to the invention, theprocesses using these solutions and, more particularly, the elementstreated according to the invention are of use for diagnostic,genotyping, high-throughput screening and quality control applications,or for detecting the presence of genetically modified organisms orpathogens in a product, or detecting the presence of dangerous materialin an object or device.

The invention is also particularly advantageous for “hybridization” or“affinity” techniques in which the intention is to analyze or separate,within a channel or a container, the species contained in a sample, as afunction of their respective specific affinity for ligands. Theseligands are either contained in said channel or container, or areattached at predetermined positions on the walls of said container orsaid channel. The invention makes it possible to carry out this type ofanalysis, while at the same time avoiding or minimizing the nonspecificadsorption of said species to the walls of the channel or of thecontainer, or to solid surfaces contained in said channel or in saidcontainer.

According to a preferred embodiment, this type of ligand may beassociated with the element, namely channel, container, element formingpart of the composition of said channel or container, or particles, viaa treatment with a surface treatment solution according to theinvention. In this instance, the treatment solution according to theinvention performs two functions. It reduces the nonspecific adsorptionand provides said ligands or contributes to immobilizing them at thelevel of said element.

A family of polymers which is particularly advantageous for applicationsof analysis by affinity consists of a block copolymer simultaneouslyhaving

1/ a multiplicity of polymer segments exhibiting an affinity specificfor a wall of the channel or of the container, or certain predeterminedparts of said walls, or else with certain solid surfaces present in saidchannel or of said container, such as the surfaces of particles or latexbeads, and

2/ one or more polymer segments not exhibiting affinity with said wallsor surfaces, and bearing ligands specific to certain species, theanalysis of which is desired. Said ligands may in particular beoligonucleotides, proteins, antibodies, peptides or, more generally,biological or synthetic polymers or polymer fragments.

The advantage of the invention in this application is that it keeps theligands linked to said walls or surfaces indirectly, while at the sametime maintaining said ligands at a considerable distance from thelatter. Specifically, in the context of the invention, the polymersegment(s) bearing the ligands do not exhibit any affinity for the walland are therefore pushed away from it by the steric interactions. Thepolymers according to the invention therefore enable the analytes tointeract with the ligands, without approaching the walls.

A subject of the present invention is also the use of the claimedsolution for minimizing the phenomena of adsorption or of electroosmosiswhich occur at the surface(s) of an element intended to be brought intocontact with a fluid and/or species contained in this fluid during thetransport, analysis, purification, separation or conservation of saidfluid.

The invention is particularly advantageous for the transport, analysisor conservation of a biological fluid containing or liable to becontaminated with inorganic, organic or biological products or liveorganisms.

As regards the devices, the surface treatment solutions, the process andthe components claimed are of particular use for microfluid systems,microtitration plates, “DNA chips and protein chips” and, moregenerally, all systems of transport and analysis involving highsurface/volume ratios, since they make it possible, through the optimalchoice of the various types of block within the polymers, to combineblocks exhibiting good affinity for the surface of the walls in order toobtain a long-lasting treatment, and blocks exhibiting good repulsionfor the species to be separated, whatever said species may be andwhatever the chemical nature of said component.

The following Examples are nonlimiting illustrations of the presentinvention. Examples 1 to 7 are concerned with the liquid separatingmedium aspect present invention. Examples 8 to 17 are concerned with thesurface treatment solution aspect of the invention.

Example 1 Preparation of a Functionalized PDMA Macromonomer with aMolecular Mass in the Region of 10,000, for the Preparation ofCopolymers in Accordance with the Invention

Polymerization of PDMA

The free-radical polymerization of N,N-dimethylacrylamide (DMA) isperformed in pure water. The initiator is a redox couple for which theoxidizing agent is potassium persulfate K2S2O2 (KPS) and the reducingagent is aminoethanethiol AET.HCl. The initiation reaction is:

K2S2O2+2C1−, NH3-CH2CH2-SH→2KHSO4+2C1−, HN3+-CH2CH2-S+

AET.HC1 also acts as transfer agent, which allows the chain length to becontrolled.

Procedure

0.18 mol of DMA and 200 ml of water are placed in a 500 ml three-neckedflask on which is mounted a condenser, and equipped with a nitrogeninlet device. The mixture is then stirred and heated to 29° C. with awater bath. Sparging with nitrogen is commenced. After 45 minutes, 0.61g of AET.HC1 (0.0054 mol) predissolved in 20 ml of water is added,followed by addition of 0.0018 mol of potassium persulfate (KPS)dissolved in a minimum amount of water. The mixture is stirred for 3hours. The solution is then concentrated and then freeze-dried.

To isolate the polymer, a precipitation is performed according to thefollowing procedure:

The solid obtained is redissolved in 100 ml of methanol. Thehydrochloride present is neutralized by adding 0.0054 mol of KOH (i.e.0.30 g dissolved in about 25 ml of methanol) incorporated dropwise intothe solution. The salt formed. KCl, precipitates and is extracted byfiltration. The filtrate thus recovered is concentrated and then poureddropwise into 4 liters of ether. The precipitated polymer is recoveredby filtration through a No. 4 sinter funnel. The solid is then driedunder vane-pump vacuum. The mass yield is about 50%.

The above protocol leads to an amino polymer known as “PDMA” andcorresponds to initiator/monomer ratios Ro=0.03 and Ao=0.01, in which:

Ro=[R—SH]/[NIPAM] and Ao=[KPS]/[NIPAN]

2) Modification of the Amino PDMA

The PNIPAM macromolecules synthesized contain amine functions at thechain ends, these chains originating from the initiator aminoethanethiolAET.HC1.

By reaction of the amine function with acrylic acid, a vinyl double bondis attached to the chain end according to the following reaction scheme:

Procedure:

50 ml of methylene chloride, 1.5 g of acrylic acid (0.021 mol), 9 g ofPDMA and 4.3 g of dicyclohexylcarbodiimide (DCCI) (0.021 mol) are placedin a 100 ml beaker.

The reaction medium is stirred for one hour. Since the acrylic acid isin large excess relative to the PDMA (the amount of acrylic acid isabout twenty times that of the PDMA), all the amino functions have beenmodified. The mixture is then filtered through a No. 4 sinter funnel toremove the precipitated dicyclohexylurea, the by-product resulting fromthe conversion of the DCCI. The purification is performed byprecipitation from ether.

A macromonomer PDMA-1 bearing an allyl function at the chain end is thusobtained with a mass yield of about 70%.

The average molar mass and the polydispersity of the macromonomers thusprepared, measured by SEC (steric exclusion chromatography), are of theorder of 15,000 and 2, respectively.

Example 2 Preparation of a Copolymer PCAN-PDMA)-1 with an AcrylamideSkeleton and PDMA Grafts, of Molecular Mass 1,500 kdalton

The copolymerization of amino PDMA (0.4 g) and of acrylamide (2.8 g) isperformed for 4 hours in 50 ml of water at room temperature, whiledegassing vigorously with argon. The initiator used is the redox coupleof ammonium persulfate ((NH4)2S2O8) [0.075 mol % of the amount ofmonomers]/sodium metabisulfite (Na2S2O5) (0.0225 mol % of the amount ofmonomers). The resulting copolymer is purified by precipitation fromacetone and dried under vacuum. Its molecular mass is 1,500 kdalton, andits polydispersity Mw/Mn is about 2. The degree of incorporation ofmacromonomer, measured by proton NMR, is about 6%, which corresponds toan average number of side branches on the skeleton of about 6.

On account of the free-radical polymerization method used, themacromonomers constituting the side chains are incorporated into thepolymer chain at random positions determined by chance by the collisionsbetween molecules (random distribution). This polymerization methodleads to a distribution of the molecular masses of the polymer segmentsof the skeleton between two side branches of approximately exponentialshape, and thus to polydispersities of said polymer segments of theskeleton that are largely superior to 1.8.

Example 3

Separation properties obtained for single-stranded DNA (50-500 bp sizer,Pharmacia Biotech) at 50° C. in an ABI 310 machine (Perkin-Elmer), in a100 mM Na TAPS, 2 mM EDTA, 7 M urea buffer, in various separation media.It is observed visually (FIG. 4) and more quantitatively by means of theresolution measurements (FIG. 5), that the separating medium accordingto the invention P(AM-PDMA)-2 improves the resolution relative to thesame polymer skeleton not bearing side branches (PAM. FIGS. 2 and 5),but also relative to a commercial product based on linear PDMA (POP6,FIGS. 3 and 5). The separation time is also reduced, which is anadditional advantage of the media according to the invention. It thusappears, surprisingly but beneficially, that this polymer according tothe invention which comprises a large fraction of acrylamide, and asmaller fraction of PDMA, has, on account of the particular arrangementof said fractions and of the presence of junction points thatcharacterize the invention, properties that are superior to those ofeach of said components in homopolymer form.

Example 4 Preparation of a Copolymer P(AM-PDMA)-2 Containing anAcrylamide Skeleton and PDMA Grafts, of Molecular Mass about 3,000kdalton

The preparation is identical to that described in 30 example 2, exceptfor the concentration of ((NH4)2S2O8) [0.1 mol % instead of 0.075 mol %of the amount of monomers] and of (Na2S2O5) (0.015 mol % instead of0.0225 mol % of the amount of monomers). The viscosity, presented inFIG. 6, makes it possible to evaluate the molecular mass, of about 3,000kdalton, starting from that of the p(AM-PDMA)-1, using the cubicdependence of the viscosity as a function of the molecular mass forinterlocked polymers.

Example 5 Preparation of a Copolymer P(AM-PDMA)-3 Bearing PDMA Grafts,of Molecular Mass about 30,000

In a first stage, the macromonomer of molecular mass 530,000 is preparedas described in example 1, with the exception of the ratio Ro, which isset at 0.015 instead of 0.03. This macromonomer is then polymerized withacrylamide, according to the protocol described in example 4.

Example 6

Measurement of the viscosity of 3% solutions obtained with the polymersdescribed in examples 2, 4 and 5, and also with a linear acrylamidehomopolymer. In this example, each of the polymers was introduced at arate of 3 g/100 ml into purified water (MilliQ). The viscosity of eachof the corresponding solutions was measured on a Brookfield DV3cone-plate rheometer run by the Rheocalc software (Sodexim, Muizon, F).The shear rate selected is 10 (I/s) for a temperature gradient of 1° C.per minute. It is observed in FIG. 6 that the copolymers according tothe invention have no thermosensitive nature (their viscosity shows asmall and uniform decrease with temperature), and a moderate viscosity.It is also observed that the structure and properties of the copolymerscan be varied by controlling the polymerization conditions.

Example 7

Electrophoretic separations of single-stranded DNA fragments, inseparating media according to the invention based on copolymersdescribed in examples 2, 4 and 5, and, for comparative purposes, inlinear polyacrylamide (LPA) and the commercial separating medium “POP5”(Applied Biosystems). The separation conditions are identical to thoseof example 4, with the exception of the sample, a “sizer” of 100 to1,500 bases (BioVentures, USA). It is observed in FIG. 7 that in therange that is most advantageous for sequencing (fragment size 600 to1,000), the media based on copolymers according to the invention, inparticular those corresponding to a mass concentration in the separatingmedium of 3%, lead to a resolution that is markedly superior to thatobtained with the polymers of the prior art. Considering that aresolution of the order of 0.3 to 0.5 is sufficient to sequence DNA towithin one base, the media according to the invention should allowreading lengths of greater than 800 bases.

Example 8 Preparation of a Functionalized PNIPAM Macromonomer of AverageMolecular Mass in the Region of 10,000 for the Purpose of PreparingCopolymers in Accordance with the Invention

1) Polymerization of the NIPAM

The radical polymerization of the NIPAM is carried out in pure water.The initiator is a redox couple in which the oxidant is potassiumpersulfate, K2S2O8 (KPS), and the reducing agent is aminoethanethiol(AET).HCl. The initiating reaction is:

K2S2O8+2Cl−, NH3+-CH2CH2-SH→2KHSO4+2Cl−, HN3+-CH2-CH2-S.

The AET.HCl also plays the role of a transfer agent, which makes itpossible to control the length of the chains.

Procedure

20 g of NIPAM (0.18 mol) and 200 ml of water are introduced into a 500ml three-necked flask surmounted by a cooling apparatus and equippedwith a nitrogen inlet device. The mixture is then stirred and heated to29° C. with a water bath. The sparging of nitrogen is begun. After 45minutes, 0.602 g of AET.HCl (0.0054 mol) dissolved beforehand in 20 mlof water are added, followed by 0.478 g of potassium persulfate (KPS)dissolved in a minimum amount of water. The mixture is maintained withstirring for 3 hours. Next, the solution is concentrated and thanlyophilized.

To isolate the polymer a precipitation is performed according to thefollowing procedure:

The solid obtained is redissolved in 100 ml of methanol. Thehydrochloride present is neutralized by adding 0.0054 mol of KOH (i.e.0.302 g dissolved in approximately 25 ml of methanol) incorporateddropwise in the solution. The salt formed, KCl, precipitates and isextracted by filtration. The filtrate thus recovered is concentrated andthen poured dropwise into 4 liters of ether. The polymer precipitatesand is recovered by filtration over sintered glass No. 4. The solid isthen dried under vacuum by a vane pump. The mass yield is of the orderof 50%.

The above protocol produces an aminated polymer name “PNIPAM-A-C”, andcorresponds to initiator-monomer ratios Ro=0.03 and Ao=0.01, where:

Ro=[R—SH]/[NIPAM] and Ao=[KPS]/[NIPAM].

2) Modification of the Aminated PNIPAM, PNIPAM-A-C

The PNIPAM macromolecules synthesized have amine functional groups atthe end of the chains, these functional groups originating from theinitiator aminoethanethiol, AET.HCl.

By reacting the amine functional group on acrylic acid, a vinyl doublebond is attached to the end of the chain according to the followingreaction scheme:

Procedure:

50 ml of methylene chloride, 1.5 g of acrylic acid (0.021 mol), 9 g ofPNIPAM and 4.3 g of dicyclohexylcarbodiimide (DCCI) (0.021 mol) areintroduced into a 100 ml beaker.

The reaction medium is stirred for one hour. Since the acrylic acid isin great excess relative to the PNIPAM (the amount of acrylic acid isapproximately twenty times that of the PNIPAM), all of the aminofunctional groups were modified. The mixture is then filtered oversintered glass No. 4 in order to remove the dicyclohexylureaprecipitate, a byproduct resulting from the transformation of the DCCI.

The mixture is then concentrated down to 15 ml and then transferreddropwise into 200 ml of ether in order to precipitate the polymer. Themixture is filtered over sintered glass No. 4 and the solid is washedwith three times 100 ml of ether and then dried under vacuum by the vanepump overnight.

A macromonomer PNIPAM-C bearing an allyl functional group at the end ofthe chain is thus obtained, with a mass yield of the order of 70%.

The molar masses of the macromonomers thus prepared were measured by SEC(steric exclusion chromatography) in THF at 40° C., with anultrastyragel column, refractometric double detection and universalcalibration with respect to polystyrene samples. NB: I have simplifiedhere.

Other macromonomers (NIPAM) were prepared according to this protocol.They are listed in table I below and are characterized in terms ofpolydispersity and molecular weight for each of the two types of segmentand by weight.

These results show that it is possible to vary the average molecularmass of the macromonomers by varying the temperature of polymerizationand the initiator/polymer ratio Ro, the highest Ro ratios leading to thelowest molecular masses. They also show that the polydispersities of themacromonomers are high, in general greater than 2.

TABLE 1 Molecular mass PNIPAM-C PNIPAM-5 PNIPAM-M PNIPAM-10 PNIPAM-LPNIPAM-20 Preparation Ro = 0.03 23° C. Ro = 0.025 23° C. Ro = 0.02 25°C. Ro = 0.02 29° C. Ro = 0.015 Ro = 0.01 25° C. conditions 25° C. Mw(g/mol) 10,800 12,800 15,800 20,400 23,000 34,000 Average number of 200230 290 370 420 620 atoms along the chain Polydispersity 5.7 2.0 4.2 3.24.9 5 (Mw/Mn)

Example 9 Preparation of a PDMA-NIPAM Copolymer with a Comb Structureand Comprising the PNIPAM-C Prepared in Example 8, as Segments Devoid ofSignificant Affinity with the Wall, and Poly(N,N-Dimethylacrylamide)(PDMA) as Backbone Showing an Affinity with the Wall

The copolymerization of the PNIPAM-C (0.7 g) and of the DMA (2.8 g) iscarried out for 4 h in 30 ml of water at ambient temperature, withvigorous degassing with argon. The initiator used is the redox coupleammonium persulfate ((NH₄)₂SO₂O₈) (0.1 mol % of the amount ofmonomers)−sodium metabisulfite (Na₂S₂O₅) (0.03 mol % of the amount ofmonomers). The resulting copolymer is purified by ultrafiltration in a“Minitan Millipore®”, equipped with a membrane having a cutoff of30,000, and then lyophilized. The final level of incorporation of PNIPAM10, measured by proton NMR on the polymers diluted to 2 g/100 ml inheavy water (Bruker devices at 250 MHz) is 6.5%. The molecular mass,measured in water at 25° C. by steric exclusion chromatography on a“Shodex®” column with refractometric double detection and two-anglelight scattering (Precision Detector), is Mw=3,000,000, and thepolydispersity is 2. The average number of side branches along thebackbone is deduced from these values and from the molecular mass of thePNIPAM-C, and is of the order of 18.

Due to the method of radical polymerization used, the macromonomersconstituting the side chains are integrated into the polymer chain atrandom positions determined by the chance of collisions betweenmolecules (statistical distribution). This method of polymerizationleads to the form of the distribution of the molecular masses of thepolymer segments of the backbone between two side branches beingapproximately exponential, and therefore to the polydispersities of saidpolymer segments of the backbone being largely greater than 1.8.

Example 10

Preparation of a macromonomer of the PDMA type bearing an acrylicfunctional group at one end: the reaction is carried out according tothe same protocol as example 8, replacing one mole of NIPAM with onemole of DMA. The purification is carried out by precipitation from etherand then filtration.

Example 11 Preparation of Copolymer “PAM-PDMA-1”, Having an AcrylamideBackbone which does not Interact with the Wall and pDMA GraftsExhibiting Strong Affinity with Silica Walls

The copolymerization of the pDMA macromonomers prepared in example 10(0.7 g) and of acrylamide (2.8 g) is carried out for 4 h in 30 ml ofwater at ambient temperature, with vigorous degassing with argon. Theinitiator used is the redox couple ammonium persulfate ((NH₄)₂S₂O₈)(0.1% of the amount of monomers)−sodium metabisulfite (Na₂S₂O₅) (0.03mol % of the amount of monomers). The resulting copolymer is purified byultrafiltration in a “Minitan Millipore®”, equipped with a membranehaving a cutoff of 30,000, and then lyophilized. The molecular mass,measured by exclusion chromatography (conditions identical to example9), is Mw=813 kDa, and the polydispersity is 2.2. The mass proportion ofpDMA, measured by NMR, is 6.5%, which corresponds on average to 5 sidebranches along the backbone. As in example 9, the method of radicalpolymerization used leads to a high polydispersity of the polymersegments of the backbone between two side branches.

Example 12 Separation Properties Obtained for DNA (50-500 bp Sizer,Pharmacia Biotech), with and without Treatment of the Silica Capillarywith the Copolymers Prepared According to Example 9

The electropherograms are obtained at 50° C. in an ABI 310® device(Perkin-Elmer), in a 50 mM Na TAPS buffer containing 2 mM EDTA and 7 murea,

a) in acrylamide without pretreatment of the capillary (FIG. 8)

b) in acrylamide after treatment of the capillary with a commercialtriblock copolymer “pluronics F127”, BASF (FIG. 9)

c) in acrylamide after treatment of the capillary with a commercial combcopolymer having a hydroxyethylcellulose backbone bearing short-chainalkyl functional groups (Natrosol Plus 331, AquaIon) (FIG. 10)

d) in acrylamide after treatment with the copolymer PDMA-NIPAM preparedaccording to example 9 (FIG. 11)

e) in acrylamide with addition of 0.5% of the copolymer PDMA-NIPAMprepared according to example 9, to the separation medium (FIG. 12).

It is noted that the use of copolymers according to the inventionconsiderably improves the sharpness of the peaks, whether this is in theform of treatment of the capillary before separation (FIGS. 10 and 11),or in the form of addition to the separation medium itself (FIG. 12).This augmentation, which is very marked with respect to the nontreatedcapillary (FIG. 8), is also significant with respect to a capillarytreated with a commercial block copolymer which does not have theminimum number of polymer segments which characterize the invention(FIG. 9). Finally, it is also noted that the copolymers exhibiting sidebranches of high molecular mass and irregular length (FIGS. 11 and 12)lead to better separations than those exhibiting branches which are oflow molecular mass and monodisperse (FIG. 10).

FIG. 14 represents the extrapolated resolution between peaks differingby one base, evaluated by interpolation from the results of the “Sizer500”. It is once again noted that this resolution is improved by thepolymers according to the invention.

Example 13 Separation Properties Obtained for DNA (50-500 bp Sizer,Pharmacia Biotech), at 50° C. in an ABI 310 Device (Perkin-Elmer), in a50 mM Na TAPS Buffer Containing 2 mM EDTA and 7 M Urea

These properties are evaluated according to two variants:

a) in acrylamide with addition of 0.5% of the copolymer PAM-PDMA-1prepared according to example 11, to the separation medium (FIG. 13a );

b) in acrylamide after treatment with the copolymer PAM-PDMA-1 preparedaccording to example 11 (FIG. 13b ).

Compared with FIG. 8, it is once again noted that the use of this othercopolymer according to the invention considerably improves the sharpnessof the peaks, whether this is in the form of treatment of the capillarybefore separation or in the form of addition to the separation mediumitself. This increase in performance is found in the measurement of theresolution, FIG. 14.

This sharpness of peaks is attributed to the property of the processesaccording to the invention, which makes it possible to reduceinteraction of the analytes with the wall. The better performancesobtained with the copolymers exhibiting polymer segments of high andirregular molecular mass are attributed to the formation of a thick and“flexible” adsorbed layer. Such a layer would make it possible to pushthe analytes away from the wall, while at the same time remaining veryswollen with water and therefore relatively unlikely to give specificinteractions with these analytes.

Example 14 Use of a Surface Treatment Solution According to theInvention in a Microfluid Cell

A microfluid cell comprising a channel 20 μm thick and 100 μm wide isprepared with polydimethylsiloxane, as described in Ocvirk et al.,Electrophoresis, 21, 107 (2000). The walls of the channel are treated byincubation for 30 min, a/ with a solution containing 3% of “PluronicsF127” and b/ with a solution containing 3% of polymers according to theinvention PDMA-NIPAM, prepared according to example 9. In both cases,the channel is rinsed, then filled with a solution of magnetic particlesand subjected to a magnetic field of 60 mTestla, as described in Mayeret al., Mat. Res. Soc. Symp. Proc. 463, 57 (1998). The presence ofelectroosmosis is tested by the movement of the magnetic particles, inan electric field of 20 V/cm. For the capillary treated with thePluronics, this displacement appears after the field has been appliedfor 2 to 3 min. For the capillary treated with the polymer according tothe invention, there is still no observable displacement after the fieldhas been applied for 3 hours. The copolymer additives according to theinvention thus provide the means for better control of the transport ofa fluid or of species contained in this fluid, in microfluid channelswhich are varied in nature.

Example 15 Preparation of a Copolymer P(AM-PDMA)-2 Having an AcrylamideBackbone and PDMA Grafts, of Molecular Mass Approximately 3,000 kDalton

The preparation is identical to that described in example 11, except forthe concentration of ((NH₄)₂S₂O₈) [0.1 mol % instead of 0.075 mol % ofthe amount of monomers] and of (Na₂S₂O₅) (0.015 mol % instead of 0.0225mol % of the amount of monomers). The viscosity, given in FIG. 13, makesit possible to evaluate the molecular mass, which is of the order of3,000 kDalton, from that of the p(AM-PDMA)-1, using the cubic dependencyof the viscosity as a function of the molecular mass for entangledpolymers.

Example 16 Preparation of a Copolymer P(AM-PDMA)-3 Bearing PDMA Grafts,of Molecular Mass Approximately 30,000

First, the macromonomer of molecular mass 30,000 is prepared asdescribed in example 6, except for the Ro ratio, which is set at 0.015instead of 0.03. This macromonomer is then polymerized with acrylamide,according to the protocol described in example 16.

Example 17 Evaluation of the Performances of Separation MediaIncorporating a Copolymer in Accordance with the Invention

The polymers added in a proportion of 0.5% are:

-   -   poly(AM-PDMA)-1 prepared according to example 11,    -   poly(AM-PDMA)-2 prepared according to example 15,    -   poly(AM-PDMA)-3 prepared according to example 16,    -   poly(DMA-PNIPAM) prepared according to example 9, and    -   the linear PDMA homopolymer which represents the comparative        test.

The results obtained are given in FIG. 15.

It is noted that the copolymers according to the invention produceperformances comparable to or greater than those of the PDMAhomopolymer, despite a much smaller fraction of monomers exhibiting astrong affinity for the wall. It is also noted that the polymers ofhigher molecular mass (poly(AM-PDMA)-2), and also those in which thegrafts are of higher molecular mass (poly(AM-PDMA-3), produce the bestresolution. On the other hand, the most hydrophobic polymer(poly(PDMA-NIPAM) produces the poorest resolution. In the particularcase of poly(AM-PDMA)-2, 10 consecutive tests were carried out withoutintermediate regeneration of the walls of the channel.

Advantageously, no decrease in performances is observed.

It is important to note that the linear PDMA homopolymer does not makeit possible to obtain such results.

1. An aqueous liquid medium for analyzing, purifying or separatingspecies inside an element comprising walls or for treating the walls ofsaid element, comprising: at least one polymer having several polymersegments that differ in chemical or topological nature, said at leastone polymer being one of (a) an irregular block copolymer and (b) anirregular comb polymer having a polydispersity of at least 1.5 for allpolymer segments of at least one type of chemical or topological natureand having side branches with a molecular mass greater than 1500; and aviscosity that does not vary by a factor of 2 or more over a temperaturerange of 20° C. or less between a temperature that is 10° C. higher thanthe solidification point of said medium and a temperature that is 10° C.lower than the boiling point of said medium, wherein, said at least onepolymer has an average of at least three junction points establishedbetween polymer segments that differ in chemical or topological nature.